The objective of this SERCA-K01 application is to advance the scientific and professional development of the candidate, Dr. Sherry Shu, BVM, PhD, towards an independent investigator position in the fields of cell biology, viral pathogenesis and antiretroviral drug discovery. Dr. Shu is a veterinarian with more than ten years of research experience applying cell and animal models to the study of cancer and infectious diseases. Her previous and current work has led to 24 peer-reviewed publications to date. Dr. Shu's long term career goal is obtain an independent faculty position at a major research university, applying both cell and animal models to better understand infectious disease processes and drug mechanisms. Regular interactions with her Mentor, Dr. Thomas E. Smithgall, and a mentoring team of three senior faculty members with complementary research expertise, will enable her to reach this goal. Research Plan: This 5-year research project is designed to study the function of HIV-1 Nef dimerization in cells and in vivo. This viral accessory protein facilitates HIV-1 replication, immune evasion and AIDS progression by hijacking host cell pathways involved in protein trafficking and cell surface receptor expression. Our laboratory has shown that Nef dimerization is essential for Nef functions. Mutations and small molecules disrupting Nef dimerization in cells reduce both Nef-induced CD4 downregulation and HIV-1 infectivity. These studies implicate Nef dimerization in these functions and support the development of Nef dimerization inhibitors as a new therapeutic approach. In this proposed study. Dr. Shu will explore the mechanisms by which dimerization controls these Nef-dependent events, and to test the role of dimerization in Nef function in a humanized mouse model of AIDS for the first time. These goals will be pursued with the following Specific Aims: Aim 1: Investigat the mechanisms by which Nef dimers regulate CD4 downregulation and HIV infectivity. We will test the hypothesis that Nef dimerization is essential for recruitment of host proteins to initiate CD4 downregulation and to enhance HIV-1 infectivity. Specifically, we will (1) test whether Nef dimers are required for recruitment of key binding partners implicated in CD4 downregulation and infectivity, including AP2, -COP, Dyn2, and Nef-associated kinases, (2) determine the functions of Nef dimers during CD4 endocytosis and degradation, and (3) investigate whether Nef dimers induce HIV-1 infectivity by reducing the incorporation of Env glycoprotein into the budding virions. We will address these important questions using both genetic and pharmacological approaches in parallel. Aim 2: Evaluate the requirement for Nef dimerization in HIV-1 infectivity and the immune response in vivo using a humanized mouse model of AIDS. Humanized mice represent a breakthrough model to study HIV infection and immune responses. Importantly, humanized mice infected with Nef-deleted HIV-1 exhibit dramatically lower viral loads and substantially less T cell depletion than mice infected with wild-type virus. We will test the hypotheses that dimerization is required for Nef function in vivo, and that the dimerization interface serves as a druggable target for small molecule Nef inhibitors. First, we will compare viral replication and CD4+ T cell depletion following infection of humanized mice with HIV-1 expressing wild-type vs. dimerization- defective Nef. Second, we will determine the effect of small molecule inhibitors of Nef dimerization on wild- type HIV replication and immune system function. Successful completion of this proposal will demonstrate the importance of dimerization to Nef-induced receptor downregulation and HIV infectivity at the cellular and whole animal levels. This project will also provide an important step towards clinical translation of Nef dimerization inhibitors as a new strategy for antiretroviral therapy. Most importantly, this proposal is specifically designed to train the Candidate in a broad range of molecular, cellular and in vivo approach to HIV/AIDS research, and generate new ideas that will serve as a springboard to independent research projects. Environment: The research career development plan for Dr. Shu includes the scientific training provided by her mentor, mentoring committee, collaborators, and consultants in a highly interactive and multi-disciplinary HIV research community at the University of Pittsburgh. This award will also allow her to attend international conferences/workshops for networking and further broaden her knowledge in viral pathogenesis, immunology, cell biology, and animal models. In addition, the University of Pittsburgh Office of Academic Career Development (OACD) offers structured training series targeting postdoctoral fellows and junior faculty members to guide them in the career development. The outstanding mentoring team, the resource-rich environment of University of Pittsburgh, and the strong institutional support for career development will prepare Dr. Shu for a successful career as an independent investigator in an academic setting.